There is a growing body of evidence that supports the idea that malignant tumors are initiated and maintained by a population of tumor cells that share similar biological properties to normal adult stem cells. Cancer stem cells (CSCs) have the ability to give rise to all cell types found in a particular cancer sample. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. A logical therapeutic angle to target these cancers is to target the stem cells themselves. This approach, however, requires the availability of screening paradigms that reflect the biology and context of the CSCs. The intricate deployments of signaling pathways that orchestrate the proliferation and differentiation of stem cells underscore the complexity of the biology of tumors. Thus, traditional high-throughput cell-based screens in cell line models are unlikely to provide the most appropriate assays as cells are out of their complex cellular context (i.e., the niche). Ideally, one would like to perform small molecule screens in whole animals to increase the chances that the small molecules identified are effective at penetrating tissues, show little toxicity, and affect the CSCs specifically. In this application, we propose to use our validated humanized-tumorogenic model in the Drosophila gut to perform a large-scale screen for small molecules that affect CSCs (Aim 1). We have developed a screening method that will allow us to screen 200,000 small molecules within two years. In addition to the screen, we will perform a number of validation studies to characterize their modes of action. Our goal is to focus on growth inhibitors that specifically affect the CSCs and not wildtype stem cells, and drugs that specifically target the Notch pathway, as it is clear that this class can be beneficial for treating mammalian colorectal cancers (Aim 2). In summary, results from these studies will allow us to identify a number of small molecules effective on CSCs, and in particular inhibitors of the Notch activity. PUBLIC HEALTH RELEVANCE: We propose to perform a large scale in vivo screen for small molecules that will be effective on Cancer Stem Cells. The screens will in particular identify inhibitor of Notch activity.